The ability to identify the gene expression differences that can account for phenotypic changes would greatly advanced our current understanding of several important biological events, including development, differentiation, transformation, and senescence. A long-term goal of this proposal is to study the gene expression differences between transformed cells and their normal counterparts. There are two outstanding issues in differential gene expression analysis. First, there is a need for methods that allow identification of a large cohort of differentially expressed genes. Second, as the differential cloning methods improve, there is a need for a mechanism that allows functional analysis of a large number of genes. In Specific Aim 1, they will concentrate on identification of differentially expressed genes, using an in vitro oral carcinogenesis model that allows independent examination of both the early and late events of oncogenic transformation. Representational difference analysis (RDA) will be improved so that a sufficiently large fraction of the gene expression differences between transformed cells and their normal counterparts can be cloned. cDNA array hybridization will be used to establish the expression profile of the cloned differentially expressed genes in other cancer cells. In Specific Aim 2, they will employ an expression cloning strategy to detect genes that can alter growth properties. The output of RDA will be used to construct an eukaryotic expression library. The complexity of such a library, compared to a standard cDNA expression library, is expected to be low and will favor the success of expression cloning. The expression cloning strategy, if successful, will significantly alter the overall approach toward differential gene expression analysis. It is certain that this investigation will lead to the development of many valuable reagents and methods for additional studies in molecular mechanisms of human epithelial cell carcinogenesis and other complex biological events.